MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2

Inflammasomes are multimeric protein complexes involved in the processing of IL-1β through Caspase-1 cleavage. NLRP3 is the most widely studied inflammasome, which has been shown to respond to a large number of both endogenous and exogenous stimuli. Although studies have begun to define basic pathways for the activation of inflammasome and have been instrumental in identifying therapeutics for inflammasome related disorders; understanding the inflammasome activation at the molecular level is still incomplete. Recent functional studies indicate that microRNAs (miRs) regulate molecular pathways and can lead to diseased states when hampered or overexpressed. Mechanisms involving the miRNA regulatory network in the activation of inflammasome and IL-1β processing is yet unknown. This report investigates the involvement of miR-133a-1 in the activation of inflammasome (NLRP3) and IL-1β production. miR-133a-1 is known to target the mitochondrial uncoupling protein 2 (UCP2). The role of UCP2 in inflammasome activation has remained elusive. To understand the role of miR-133a-1 in regulating inflammasome activation, we either overexpressed or suppressed miR-133a-1 in differentiated THP1 cells that express the NLRP3 inflammasome. Levels of Caspase-1 and IL-1β were analyzed by Western blot analysis. For the first time, we showed that overexpression of miR-133a-1 increases Caspase-1 p10 and IL-1β p17 cleavage, concurrently suppressing mitochondrial uncoupling protein 2 (UCP2). Surprisingly, our results demonstrated that miR-133A-1 controls inflammasome activation without affecting the basal expression of the individual inflammasome components NLRP3 and ASC or its immediate downstream targets proIL-1β and pro-Caspase-1. To confirm the involvement of UCP2 in the regulation of inflammasome activation, Caspase-1 p10 and IL-1β p17 cleavage in UCP2 of overexpressed and silenced THP1 cells were studied. Suppression of UCP2 by siRNA enhanced the inflammasome activity stimulated by H₂O₂ and, conversely, overexpression of UCP2 decreased the inflammasome activation. Collectively, these studies suggest that miR-133a-1 suppresses inflammasome activation via the suppression of UCP2.     

Thermally stable harpin,HrpZPss is sensitive to chemical denaturants: Probing tryptophan environment,chemical and thermal unfolding by fluorescence spectroscopy

Harpins – a group of proteins that elicit hypersensitive response (HR) in non-host plants – are secreted by certain Gram-negative plant pathogenic bacteria upon interaction with the plant. In the present study, the microenvironment and solvent accessibility of the sole tryptophan residue (Trp-167) in harpin HrpZ_Pss, secreted by Pseudomonas syringae pv. syringae, have been characterized by fluorescence spectroscopic studies. Emission λ_max of the native protein at 328 nm indicates that Trp-167 is buried in a hydrophobic region in the interior of the protein matrix. Significant quenching (53%) was seen with the neutral quencher, acrylamide at 0.5 M concentration, whereas quenching by ionic quenchers, I⁻ (∼10%) and Cs⁺ (negligible) was considerably lower. In the presence of 6.0 M guanidine hydrochloride (GdnHCl) the emission λ_max shifted to 350.5 nm, and quenching by both neutral and ionic quenchers increased significantly, suggesting complete exposure of the indole side chain to the aqueous medium. Fluorescence studies on the thermal unfolding of HrpZ_Pss are fully consistent with a complex thermal unfolding process and high thermal stability of this protein, inferred from previous differential scanning calorimetric and dynamic light scattering studies. However, the protein exhibits low resistance to chemical denaturants, with 50% unfolding seen in the presence of 1.77 M GdnHCl or 3.59 M urea. The ratio of m value, determined from linear extrapolation model, for GdnHCl and urea-induced unfolding was 1.8 and suggests the presence of hydrophobic interactions, which could possibly involve leucine zipper-like helical regions on the surface of the protein.     

Design,synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9

Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2′-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1β and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.     

The X-ray Crystal Structure of Mannose-binding Lectin-associated Serine Proteinase-3 Reveals the Structural Basis for Enzyme Inactivity Associated with the Carnevale,Mingarelli, Malpuech,and Michels (3MC) Syndrome

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.     

A Molecular Switch Governs the Interaction between the Human Complement Protease C1s and Its Substrate,Complement C4

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492–499 and 573–580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.     

Quantitative proteomics for identifying biomarkers for Rabies

Introduction

Rabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite.

Methods

In this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy.

Results and conclusion

We identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.     

Molecular dynamics studies on conformational thermodynamics of Orai1–calmodulin complex

Molecular understanding of bio-macromolecular binding is a challenging task due to large sizes of the molecules and presence of variety of interactions. Here, we study the molecular mechanism of calmodulin (CaM) binding to Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. Although experimental observations indicate that Orai1 binds to the C-terminal of Ca²⁺-loaded CaM, it is not decisive if N-domain of CaM interacts with Orai1. We address the issue of interaction of different domains of CaM with Orai1 using conformational thermodynamic changes, computed from histograms of dihedral angles over simulated trajectories of CaM, CaM-binding domain of Orai1 and complexes of CaM with Orai1. The changes for all residues of both C and N terminal domains of CaM upon Orai1 binding are compared. Our analysis shows that Orai1binds to both C-terminal and N-terminal domains of CaM, indicating 1:2 stoichiometry. The Orai1 binding to N-terminal domain of CaM is less stable than that to the C-terminal domain. The binding residues are primarily hydrophobic. These observations are in qualitative agreement to the experiments. The conformational thermodynamic changes thus provide a useful computational tool to provide atomic details of interactions in bio-macromolecular binding.     

MOTOR: Model assisted software for NMR structure determination

Eukaryotic proteins with important biological function can be partially unstructured, conformational flexible, or heterogenic. Crystallization trials often fail for such proteins. In NMR spectroscopy, parts of the polypeptide chain undergoing dynamics in unfavorable time regimes cannot be observed. De novo NMR structure determination is seriously hampered when missing signals lead to an incomplete chemical shift assignment resulting in an information content of the NOE data insufficient to determine the structure ab initio. We developed a new protein structure determination strategy for such cases based on a novel NOE assignment strategy utilizing a number of model structures but no explicit reference structure as it is used for bootstrapping like algorithms. The software distinguishes in detail between consistent and mutually exclusive pairs of possible NOE assignments on the basis of different precision levels of measured chemical shifts searching for a set of maximum number of consistent NOE assignments in agreement with 3D space. Validation of the method using the structure of the low molecular‐weight‐protein tyrosine phosphatase A (MptpA) showed robust results utilizing protein structures with 30–45% sequence identity and 70% of the chemical shift assignments. About 60% of the resonance assignments are sufficient to identify those structural models with highest conformational similarity to the real structure. The software was benchmarked by de novo solution structures of fibroblast growth factor 21 (FGF21) and the extracellular fibroblast growth factor receptor domain FGFR4 D2, which both failed in crystallization trials and in classical NMR structure determination. Proteins 2013; 81:2007–2022. © 2013 Wiley Periodicals, Inc.     

Synthesis and anti-acetylcholinesterase properties of novel β- and γ-substituted alkoxy organophosphonates

Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at which post-inhibitory reactivation or aging phenomena occurs. In this report, we prepared a panel of β-substituted ethoxy and γ-substituted propoxy phosphonoesters of the type p-NO₂PhO-P(X)(R)[(O(CH₂)_nZ] (R = Me, Et; X = O, S; n = 2, 3; Z = halogen, OTs) and examined the inhibition of three AChEs by select structures in the panel. The β-fluoroethoxy methylphosphonate analog (R = Me, Z = F, n = 2) was the most potent anti-AChE compound comparable (k_i ～6 × 10⁶ M^?1 min^?1) to paraoxon against EEAChE. Analogs with Z = Br, I, or OTs were weak inhibitors of the AChEs, and methyl phosphonates (R = Me) were more potent than the corresponding ethyl phosphonates (R = Et). As expected, analogs with a thionate linkage (PS) were poor inhibitors of the AChEs.     

Cloning,expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus

Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: {"type":"entrez-nucleotide","attrs":{"text":"JN645812","term_id":"374095279"}}JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose K_m 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.